The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.
The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.
Since 1973 the kidney transplant model in mice has been a valuable research tool, but technical issues have hampered its widespread use. Over the years several papers have been published detailing improvements/refinements to this procedure. As a model of primarily vascularized solid organ transplantation this procedure is probably second only to the heterotopic heart transplant model which was also devised by the Russell laboratory in 1973 6. Both models lend themselves to research into allogeneic rejection responses, the development of delayed graft function and ischemia reperfusion injury.
One of the most common issues to be reported with kidney transplantation is the relatively high incidence of arterial thrombosis 4,5,7 which we also experienced in our laboratory. Therefore we set out to perform a literature review of thrombus formation and possibly find the cause of this technical issue and to also devise a possible solution. The most likely cause of thrombosis is the somewhat tortuous path the blood takes from the recipient aorta, into the donor renal aorta then on to the donor renal artery. This path causes turbulence in the renal artery which can lead to platelet activation and thrombus formation. Based on the recent observations and a search of relevant literature 8-14 we came up with a new technique that has reduced thrombosis to 0%.
The technique described here varies from previously reported techniques in the formation of an arterial heel-and-toe cuff which facilities improved blood flow and significantly reduces thrombus formation. The cuff is formed by dividing the infra-renal aorta across the face of the renal arterial ostium at an angle less than 45o to the longitudinal axis of the aorta (Figure 1A & 1B). This results in a cuff approximately 2mm in length. A venous Carrel patch is formed by transecting the renal vein into the IVC thereby increasing the diameter of the cuff. The infra-renal donor abdominal aorta heel-and-toe cuff is end-to-side anastomosed to the recipient abdominal aorta and the donor renal vein/IVC patch is end-to-side anastomosed to the recipient abdominal inferior vena cava (IVC). The ureter is then introduced into and anchored to the bladder as described by Han et al 3.
For this study untreated transplants with warm ischemia times only (i.e., no cold ischemia) are compared. In this case warm ischemia refers to the time from the cessation of blood flow through the donor kidney (step 1.11 below) and reperfusion of the graft in the recipient (step 2.11 below). Cold ischemia refers to the time that the kidney is not perfused and is kept in cold storage until the beginning of the implant procedure.
All mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were housed under pathogen free conditions at the University of Colorado Denver, Barbara Davis Center Animal Facility according to NIH Guidelines and with approval of the University of Colorado Denver IACUC.
1. Donor Kidney Harvest
2. Kidney Implant Technique
3. Contralateral Nephrectomy
4. Graft Assessment
This surgical technique allows for either simple graft survival/rejection studies, or quite complex experimental protocols. In the figures below we demonstrate the advantages of using this improved arterial anastomosis technique. Using this technique we have significantly reduced the incidence of arterial thrombosis from 35% to 0% thus increasing productivity. We have used this technique for over one year with the same 0% thrombosis result maintained. Figure 1 describes the method for the formation of the arterial heel-and-toe cuff which is the basis of this new technique. This cuff provides for a longer anastomosis and a straighter blood flow path into the kidney. Both of these points result in significantly lessened turbulence thereby reducing the likelihood of platelet activation and thrombus formation.
Figure 1. Schematic representation of the preparation of the donor cuff and the resulting arterial anastomosis. (A) A line diagram depicting the formation of the arterial heel-and-toe cuff. The cuff is formed by dividing the abdominal aorta obliquely across the renal arterial ostium at an angle less than 45o to the longitudinal axis of the aorta. (B) A simplified diagram showing how the infra-renal aorta is divided to form the cuff. (C) The cuff depicting the resulting lumen. (D) The completed anastomosis (venous and ureteric structures not shown for clarity) results in a large cross-sectional lumen and a straight blood flow path.
Figure 2. Immunohistochemical analysis. (A) Periodic Acid-Schiff stained section of a control non-transplanted kidney. (B) A syngeneic kidney transplant at POD 8 using the Revised technique demonstrates normal proximal tubular cells, which are cuboidal, with a clear cytoplasm and a round light nucleus in the middle of the cell. There is evidence of mild brush border injury (arrows) but the majority of brush borders are regular and well preserved. Magnification: 400x.
Table 1. Comparison of thrombosis incidence and warm ischemia times between the two techniques. All values are expressed as mean ± SD. For single comparisons, normally distributed data are evaluated using unpaired, two-tailed Student t tests, and non-normally distributed data are analyzed by the nonparametric unpaired Mann-Whitney U test. P values of less than 0.05 are considered statistically significant.
Mastering this transplant technique is difficult, but once accomplished it is a very powerful research tool. The patient surgeon/researcher will be rewarded by attention to detail and consistency of technique, which is the key to mastering any surgical procedure, even more so in small animal models. The technical difficulties of mastering the mouse kidney transplant are many folds, and it is highly probable that experience in other small animal transplant models must be gained before tackling this procedure.
It is important to ensure that the anastomoses are “clean”; i.e., the opposing vascular walls must not be caught when placing stitches in order to maintain a patent lumen. Otherwise there will be significant constriction to flow that will more than likely result in a failed graft and in extreme cases lead to hind-limb paralysis. It is also vitally important that full thickness passes of the suture needle including the vascular adventitia and the intima are achieved as this results in proper evertion of the edges ensuring that there is intima-to-intima contact which aids in sealing and healing of the anastomoses. While a hemostatic clotting agent can be useful for reducing leaks, we recommend that a surgeon instead rely upon good technique.
Also attention must be paid to ensuring the tension of the anastomotic suture lines is optimal, too loose and there will be irreversible leaking, to tight and reduced flow will result. If on the arterial side this will result in poor perfusion of the graft, if on the venous side a congested kidney will result. Attaining this correct tension is a matter of practice and experience with other small animal microvascular models will be of great assistance. Above all consistency of technique and unwavering attention to detail will yield excellent mouse and graft survival, and kidney transplant function.
The limitations of this technique are governed only by the uses which can be applied by the investigator. As with any microvascular procedure, so long as good technique is observed the results should be reproducible.
The incidence of arterial thrombosis using this technique has been drastically reduced. This results in at least 1/3 of all kidney transplants being converted from potential technical failures to usable data, resulting in reduced costs and increased productivity.
As a fully vascularized, orthotopic transplant model the future applications will include rejection/tolerance studies, the phenomenon of delayed graft function and investigations into the mechanisms of ischemia/reperfusion injury.
The authors have nothing to disclose.
This work was supported in part by 1R03DK096151. We acknowledge the UAB-UCSD O’Brien Center (NIH P30 DK079337) for providing initial technical assistance with setting up this model.
Instrument | Roboz # | Fine Science Tools # | Arosurgical # |
Straight micro-dissecting forcep #5 | RS-5015 | 11295-51 | |
Curved micro-dissecting forcep #7 | RS-5047 | 11297-00 | |
Curved serrated forcep | RS-5137 | 11052-10 | |
Vannas micro-dissecting scissors, short | RS-5610 | 09.140.08 | |
Micro-dissecting scissors, straight, sharp, long | 11.602.11 | ||
Micro spring handle needle holder | 11.549.15 | ||
Straight mosquito forcep | 91308-12 | ||
Micro-dissecting scissors, straight, blunt | RS-5962 | 14078-10 | |
Micro-dissecting scissors, curved, blunt | RS-5981 | 14079-10 | |
Micro retractor | RS-6540 | ||
Instrument tray, 10” x 6 ½” x ¾” | RT-1350S | ||
Silk suture, 5/0, 22.5m spool | 18020-50 | ||
Suture | |||
10/0 nylon | T4A10Q07 | ||
5/0 silk | E19A05N | ||
Gloves | Drapes | ||
Biogel from Medex Supply | Precept, #64-9012-9 | ||
Syringes | Cotton applicators | ||
B-D 1cc insulin, #329424 | Fisher-brand, #23-400-100 | ||
Povidone-Iodine swabs | |||
PDI, #B40600 | |||
4/0 Cotton ties | |||
Domestic cotton autoclaved with instruments |